Microbiological systems

ABSTRACT

Microbiological systems for the automation of a microbiological laboratory including apparatus and methods for dilution of a microbiological sample, distribution of the sample to utilization devices, and application of the microbiological sample onto a growth medium in a sterile condition. In one embodiment, a fluid amplifier device is utilized for dilution of the microbiological sample and a fluid amplifier is also utilized for distribution of the diluted sample to a receiving well. In another embodiment, the microbiological sample is linearly applied to an elongated petri dish for subsequent incubation. Appropriate elongated petri dishes are described for use with such linear applications. A piston-cylinder arrangement is also provided for serial dilution of the samples.

BACKGROUND OF THE INVENTION

This invention relates generally to microbiological systems, and moreparticularly to specific methods and apparatus for use in amicrobiological laboratory for miniaturization and automation ofmicrobiological procedures.

Microbiology laboratories are generally equipped with numerous apparatusfor providing serial dilutions of the microbiological sample,distribution equipment for applying the diluted samples to appropriategrowth medium, incubation equipment where the micro-organisms arepermitted to grow on the medium, and various testing equipment toanalyze the results of the growth. The various apparatus must generallybe maintained in a sterile atmosphere to prevent cross contaminationfrom other microbiological organisms. Although various automatedequipment has been suggested for use in the microbiological laboratory,these equipment have been of extremely large scale and have been oflimited value since many of them can perform only one small aspect ofthe microbiological laboratory. Much of the laboratory still relies uponindividualized manual effort. As a result, the size of themicrobiological laboratory is generally extensive, the number ofpersonnel is quite high, and the cost of any anaylsis is excessive.

One of the reasons for the difficulty in miniaturizing and automatingthe microbiological laboratory involves the problem of providingappropriate dilutions of the microbiological sample. Generally, someform of serial dilution is required, typically a log distribution, inapplying the sample onto a growth medium. This is generally achieved bythe dilution of a sample and application of the diluted sample by meansof a loop onto a growth medium utilizing a serpentine streaking onto aPetri dish. The automated equipment heretofore provided tried tomechanically duplicate the serpentine streaking onto the Petri dish. Asa result, the size of the equipment needed was quite large and the speedwas relatively slow.

A further problem with miniaturizing and automating the microbiologicallaboratory concerned the distribution of diluted samples into receivingwells. Although small receiving wells could be achieved on a singlearray, the problem faced was how to apply the exact amount of dilutedsample to each receiving well. Various prior art apparatus utilized aninjection principal which filled the receiving wells in a stepwisemanner. This process was slow and again required large size equipment.

Numerous other problems unique to the microbiological laboratory haveprevented the automation and miniaturization of the equipment needed.For example, the need for maintaining the equipment in a sterileatmosphere provided specific restraint which prevented directapplication of automatic equipment from other laboratories into themicrobiological field. Additionally, the necessity for providing theserial dilution of the sample provides an increased time and spaceproblem not necessarily required in other scientific laboratory work.Accordingly, while other fields of medicine, such as biochemistry, haveadvanced to the state of automation and miniaturization, themicrobiological laboratory has yet to achieve the advanced state ofprogress which has been available in related fields of medicine.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a microbiologicaldevice for use in dilution or distribution of a microbiological sample.At least one fluid amplifier is utilized wherein the fluid amplifierincludes a control chamber, an inlet port for feeding a working fluidinto the control chamber, two fluid outlet ports for selectivelydischarging the contents of the control chamber, and a control portmeans fluidly coupled to the control chamber for utilizing a controlfluid to direct at least a portion of the working fluid to the selectedoutlet port. A supply means provides the working fluid in the form of amicrobiological sample to the inlet port. A utilization means receivesthe working fluid from the selected outlet port and can utilize it formicrobiological use.

When utilized as a dilution device, the supply means includes two inputsfluidly coupled to the fluid amplifier inlet ports whereby the supplymeans provides the microbiological sample to one of the inputs andprovides a fluid diluent to the other of the inputs. The control chamberserves as a mixing chamber to dilute the sample. In addition to thesingle fluid amplifier, a plurality of such amplifiers can be connectedin series to provide the desired serial dilution.

When utilized as a distribution device, there is further included aselector which is coupled to the control port means for initiallydirecting all of the sample to one outlet and subsequently switching thesample to the other outlet to discharge a constant quantity of thesample to a receiving well. A plurality of the fluid amplifiers can beconnected in series to provide substantially equal distributions to thereceiving wells.

The dilution and/or distribution devices can be included directly intothe cover portion of a large Petri dish wherein the lower portion has anarray of receiving wells to receive the serially diluted samplestherein.

A further embodiment of the microbiological system of the presentinvention includes a method and apparatus for linear streaking. Theapparatus operates in a sterile condition and conveys an elongated trayhaving growth medium thereon to an application stage. At the applicationstage a microbiological sample is linearly applied onto the growthmedium in the elongated tray. The elongated tray is removed from theapplication stage and a cover is placed onto the elongated tray.

A linear Petri dish is also provided for use in the linear streakingsystem and includes a rectangular base plate for receiving a growthmedium thereon. Side walls are formed around the base plate and a covermember is removably positioned over the side walls for permitting anindirect flow of air to the growth medium. In an embodiment of theinvention, the side walls are retained as part of the cover member andare placed over the rectangular base plate.

The method of linear streaking is also covered whereby a plurality ofelongated microbiological trays having growth medium thereon areutilized. The trays are arranged in a predetermined sequence and themicrobiological sample is linearly applied to the growth medium in thetrays whereby the contents of each successive tray has a differentrelationship between the sample and the growth medium as compared to thepreceeding tray.

In order to achieve serial dilution, the microbiological system can alsoinclude a piston and cylinder device with the piston movable in thecylinder by means of a rod coupled to the piston and extending throughcylinder. An outlet port is provided at one end of the cylinder. A firstinlet is provided intermediate the outlet and the piston for receiving amicrobiological sample. A second inlet on the opposite side of thepiston receives diluent. A one-way passage is provided from one side ofthe piston to the other. In this manner, reciprocation of the pistonprovides a serially diluted sample at the output of the cylinder.

It is therefore an object of the present invention to provide improvedmicrobiological devices, as described above, which can be utilized aspart of a microbiological system in automating a microbiologicallaboratory and which avoids the aforementioned problems of the priorart.

A further object of the present invention is to provide amicrobiological device utilizing a fluid amplifier for controlling theflow of the microbiological sample.

Yet another object of the present invention is to provide amicrobiological dilution device utilizing a fluid amplifier forcontrolling the dilution of the microbiological sample.

A further object of the present invention is to provide amicrobiological device providing serial dilution of a microbiologicalsample and utilizing a plurality of serially connected fluid amplifiers.

Yet a further object of the present invention is to provide amicrobiological distribution device utilizing a fluid amplifier forproviding distribution of a microbiological sample.

Still another object of the present invention is to provide amicrobiological distribution device having a plurality of seriallyconnected fluid amplifiers for providing substantially equaldistributions of a microbiological sample to receiving wells.

A further object of the present invention is to provide amicrobiological device such as a large Petri dish having an array ofreceiving wells in the base portion and having a dilution and/ordistribution mechanism in the cover portion.

Yet another object of the present invention is to provide for the use ofa fluid amplifier in a microbiological device wherein the fluid sentthrough the fluid amplifier is utilized as the working fluid.

A further object of the present invention is to provide a method oflinear streaking of Petri dishes.

Yet a further object of the present invention is to provide for thesimulation of the standard serpentine streaking by utilizing linearsegments for the application of a microbiological sample.

A further object of the present invention is to provide an apparatus forlinear streaking of microbiological samples onto a growth medium.

Another object of the present invention is to provide a linear Petridish for use in the linear streaking of microbiological samples.

Still a further object of the present invention is to provide a methodof linear streaking.

Another object of the present invention is to provide a piston andcylinder arrangement for use as a serial dilution device.

These and other objects, features and advantages of the invention will,in part, be pointed out with particularity, and will, in part, becomeobvious from the following more detailed description of the presentinvention taken in conjunction with the accompanying drawings, whichform an integral part thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In the various figures of the drawing, like reference charactersdesignate like parts.

In the drawings:

FIG. 1 is a schematic drawing of a fluid amplifier utilized as a singlestage of dilution of a microbiological sample;

FIG. 2 is a schematic drawing showing a series of interconnected fluidamplifiers for use in serial dilution of a microbiological sample;

FIG. 3 is a schematic drawing of a series of interconnected fluidamplifiers utilized for the distribution of microbiological samples;

FIG. 4 is a schematic drawing showing a plurality of seriallyinterconnected paths of fluid amplifiers as incorporated in the cover ofa Petri dish for use in the distribution of microbiological samples toreceiving wells;

FIG. 5 shows a sequence of linear Petri dishes, in accordance with thepresent invention;

FIG. 6 schematically shows a top view of a sequence of interconnectedlinear Petri dishes coupled in a serpentine fashion;

FIG. 7 schematically shows apparatus for linear streaking in accordancewith the present invention;

FIG. 8 shows a sectional elevational view of the embodiment of a linearPetri dish in accordance with the present invention;

FIG. 9 shows a fragmentary sectional view taken along line 9--9 of FIG.8;

FIG. 10 is a schematic isometric drawing showing another embodiment ofthe linear Petri dish in accordance with the present invention, and

FIG. 11 is a schematic elevational view of a piston and cylinderarrangement utilized for serial dilution in accordance with the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to provide automation and miniaturization to themicrobiological laboratory, it is necessary to provide for variousmicrobiological systems that can be easily manufactured, occupy littlespace, are reliable, and can provide exact, reproducible results. Inmost cases, apparatus introduced into the microbiological laboratorywere variations of corresponding equipment utilized in other medicallaboratories. However, it has been found that a particular device canhave unique applicability to the microbiological laboratory even thoughheretofore its use has been associated with a complete differenttechnology and its applications thus far have been completely foreign tomedical use.

In the field of electronics there has recently developed a technologyutilizing fluid logic elements. Such elements are broadly designated as"fluid amplifiers" and can be combined to provide various logic elementsto simulate electronic circuits. The logic functions are carried out byan interaction between jets of air or liquid and the device contains nomoving parts or electronic circuits.

Typically, a fluid amplifier includes a control chamber with an inletport for feeding a fluid into the control chamber. Two fluid outletports are provided for discharging the contents of the control chamber.Control ports are fluidly coupled to the control chamber and can directa control fluid to the control chamber. By way of example, the fluidpassing into the inlet port can be water which is controlled by a streamof air utilized in the control ports. By controlling the air pressure ateither of the control ports, the water can be selectively directed toeither of the outlet ports.

In the analog type fluid amplifier, the amount of water discharged ateach of the fluid outlet ports can be selectively controlled as desired.In a digital type fluid amplifier, the liquid is discharged from onlyone of the outlet ports which is controlled by the air stream toeffectively provide a switch between the two outlet ports.

The fluid amplifiers have been combined in series, stacked, placed inparallel, and interconnected to produce numerous mathematical logicfunctions. It has even been suggested to provide a fluid computerutilizing such fluid amplifiers. The fluid amplifier can be formed in anextremely thin plastic layer and occupies extremely little space andmany such fluid amplifiers can be stacked together occupying minimumsize and requiring inexpensive manufacturing costs.

Further information on fluid amplifiers can be obtained in numeroustextbooks, patents, and technical and trade journals. By way of examplereference is had to the textbook "Fluid Amplifiers," by Joseph M.Kirshner, McGraw-Hill Company, 1966.

The prior art utilization of fluid amplifiers used the liquid passingthrough the fluid amplifier only as an indicator of the analog ordigital result. In the case of the digital fluid amplifier, the onlyinterest was into which outlet port the liquid passed. The specificliquid utilized was of relative unimportance. The liquid itself was onlyused as a means for providing the indication. The fluid amplifier itselfwas not utilized to provide any direct action onto the fluid andheretofore the fluid itself was not used for any positive purpose initself.

It has been found, that the fluid amplifier can actually be utilized asa basic tool in a microbiological system whereby the fluid amplifier canserve as the basic element for either dilution of a microbiologicalsample and/or distribution of the sample to appropriate receiving wells.Thus, the essential concept is to utilize the fluid amplifier to actupon the fluid whereby the fluid itself becomes a working fluid which isacted upon and subsequently utilized for further testing. Thus, thepresent invention teaches to provide as the fluid in the fluid amplifiera microbiological sample which passes through the fluid amplifier and iseither diluted within the fluid amplifier by a diluent and/or can bedistributed by the fluid amplifier to appropriate receiving wells.

Referring now to FIG. 1, there is shown an embodiment utilizing thefluid amplifier to provide a single stage of dilution of amicrobiological sample. The fluid amplifier shown generally at 10,includes a control chamber 12 through which the fluid enters through aninlet port 14. Two fluid outlet ports 16, 18, are connected to thecontrol chamber for selectively discharging the contents of the controlchamber. Control ports 20, 22 are fluidly coupled to the controlchamber. A control fluid, such as air, is directed selectively throughthe control ports to direct at least a portion of the working fluid fromthe control chamber into the fluid outlet ports 16, 18.

Two inputs 24, 26 feed into the inlet port 14. A supply means 26'provides a sample input through the input 24 while a further supplymeans 28 provides a diluent to the input 26. The sample and the diluentare sent through their respective inputs and reach the control chamberwhich serves as a mixing chamber to dilute the sample with the diluent.The diluted sample is then appropriately controlled by means of thecontrol ports 20, 22 to selectively discharge a desired amount of thediluted sample through the outlet port 16 as the output O1 which can besent to receiving wells. The remainder of the diluted sample designatedas output O2 can be sent through the outlet port 18 and can bedischarged. In order to improve the mixing, baffles 30 are placed withinthe control chamber.

It is understood that the drawing shown in FIG. 1 is only schematic andthat the fluid amplifier itself would be formed within a plastic orglass medium in accordance with well known fluid amplifier technology.Furthermore, the appropriate ports, are all in accordance with the wellknown fluid amplifier technology and the air pressures would becontrolled in accordance with such known teachings. The supply means forsupplying the sample and diluent can be any of the well known supplydevices presently utilized in microbiological technology includinginjection means, pumps, nozzles, etc. The percent dilution can becontrolled by controlling the relative concentrations of the sample andthe amount of diluent to provide any dilution percentage desired. Theportion of the output can be selectively chosen as a desired percent ofthe total amount of diluted sample. The output selected can be directedto receiving wells directly or through a distribution system. Theportion discharged can be sent through additional series of dilutions bymeans of further fluid amplifiers each further diluting the sample inaccordance with desired concentration ratios.

Referring now to FIG. 2, there is shown a series of interconnected fluidamplifiers of the type described in FIG. 1 wherein the amplifiers areconnected to provide serial dilution of the initial sample. As is shownschematically in FIG. 2, there are three stages of fluid amplifiers 32,34, 36. Each fluid amplifier includes a control chamber 38 with twoinputs 40, 42 respectively feeding into an inlet port 44 at the entranceof the control chamber. Outlet ports 46, 48 can selectively dischargethe fluid from the control chamber. Control ports 50, 52 feed a controlfluid into the control chamber to selectively direct at least a portionof the fluid into the desired outlets.

One outlet 48 from each fluid amplifier feeds a distribution system. Theother outlet 46 feeds into one of the inputs of the next stage fluidamplifier.

A sample is provided from a supply means 54 at one input 40 of the firststage. The one input of each successive stage is fed with the outputfrom the previous stage. Diluent is fed to the other input of each ofthe stages. The diluent comes from a supply means 56 and can be the samesupply means for all of the stages. Thus, a common diluent supply can beutilized and fed into each of the other inputs of all stages.

The sample provided to the first stage is diluted by a desired percentby controlling the concentration of the sample and the amount ofdiluent. The amount desired for usage is controlled by the control portsand that portion is sent to the distribution system for utilization. Theremaining diluted sample passes to the next stage where additionaldiluent is provided to lower the concentration of the sample by furtherdiluting it. A portion of that further diluted sample is then selectedfor distribution with the remainder passing onto the next stage forstill further dilution. This can continue for numerous stages inaccordance with the number of dilutions and steps desired.

It is accordingly appreciated that by serially interconnecting the fluidamplifiers a serial dilution of the original sample can be achieved ateach successive stage of the fluid amplifier system. The amount selectedfor the distribution system can be identical at each stage whereby thesame amount of diluted sample of each serial dilution step can betested. The control fluid, such as air, can be appropriately selected toproduce the desired amount for the distribution system and can besimultaneously controlled for all of the stages. Baffles or similarmixing means could be included in the control chambers of each stage tofurther insure proper mixing of the sample with the diluent to achieveproper dilution at each stage.

The fluid amplifier can be utilized not only for single stage of serialdilution but could also be used as part of a distribution system fordistributing the sample itself or the diluted sample to appropriatereceiving wells, test tubes, or other receiving means. Referring now toFIG. 3, there is shown the use of the fluid amplifier in amicrobiological distribution system. A series a four fluid amplifiersare shown serially interconnected. The first fluid amplifier showngenerally at 58 includes a control chamber 60 having an inlet port 62with two outlet ports 64, 66. Control ports 68, 70 feed the controlfluid to the control chamber to selectively direct the fluid to thevarious outlet ports. One outlet port 66 of the first stage feeds theinlet port 70 of the next fluid amplifier 72. In a similar fashion, oneoutlet port of each stage feeds the inlet port of the next successivestage.

At the end of the other outlet port 64 is a discharge port 74 where themicrobiological fluid can be discharged to a receiving well or otherreceiving means. At the end of the outlet port 76 of the last fluidamplifier stage is a detector shown as the membrane switch 78 which candetect when fluid has passed through the last stage. The control fluid,such as air, is provided as the control fluid C1, C2 and is selected bymeans of the selector switch 80. The switch can be a control valve whichreceives the control fluid, such as air, and selectively sends the fluidinto the C1 ports of all the amplifiers whereby the fluid is directed tothe output 02 of the amplifiers, or can switch the control fluid to thecontrol ports C2 whereby the fluid will pass to the outputs 01 of eachamplifier.

In operation, a supply means 82 provides the microbiological sample inoriginal or diluted form to the first stage of the amplifiers. Thecontrol fluid is directed through inputs C1 whereby the sample will passto the output 02 of the first stage and from there feed into the nextstage. Continuous feeding of the sample into the distribution systemwill cause the fluid to continuously flow through the outputs 02 of thesuccessive stages until it completely fills the stages and reaches thedetector 78. The detector responding to the complete filling of allstages will signal the selector switch to switch the control fluid tothe control C2 of each stage whereby the fluid will be switched at eachstage from the outlet 02 to the outlet 01. Each outlet 01 is associatedwith a receiving well whereby the contents of each section of fluidamplifier will flow into the receiving well.

It should be appreciated, that by making the size of each of the fluidamplifiers substantially identical, a substantially identical amount ofthe sample will be distributed to each of the receiving wells.Specifically, referring to the stage 84, the fluid filling the distanced from the inlet port of the control chamber to the outlet ports will beidentical as the corresponding distance for all of the stages. Themicrobiological fluid filling that portion will be discharged into thereceiving wells. However, the amount of microbiological fluid in eachstage filling the corresponding portion will be substantially identical.Accordingly, a substantially equal amount of microbiological fluid willbe discharged by means of the distribution system shown in FIG. 3.

The size of the fluid amplifier utilized for dilution and/ordistribution is small enough whereby the series of fluid amplifiersconnected for dilution and/or distribution can be directly built intothe cover portion of a Petri dish having an array of receiving wells. Inthis manner, the microbiological sample can be sent directly into thecover portion of a Petri dish for dilution and subsequent distributionto receiving wells directly beneath the cover.

By way of example, FIG. 4 schematically shows a distribution systemusing fluid amplifiers which is built into the cover portion of a Petridish whereby the diluted sample can be fed into the cover anddistributed in equal amounts to receiving wells in the lower portion ofthe Petri dish. The Petri dish is shown generally at 86 and includes thecover portion 88 and the lower portion 90. An array of receiving wells92 are formed in the lower portion. Each receiving well can typicallyinclude a growth medium. Alternately, the lower portion can be a holderfor a series of test tubes whereby the distribution will be directlyinto the test tubes. Similarly, distribution can be into any other arrayof receiving means built into or connected to the lower base portion 90.

The upper portion 88 is shown to include three parallel paths 94, 96, 98of fluid amplifiers with four fluid amplifiers in each parallel path.Specifically referring to the path 94, the first fluid amplifier on theleft of that path has a control chamber 100 with an inlet port 102 and afirst outlet port 104 feeding into the next inlet 106 of the nextcontrol chamber 108. The second outlet of the first amplifier 110includes a discharge port 112 which is disposed over a correspondingreceiving well so that the fluid can be directed to the receiving welltherebeneath. The control ports 114, 116 are fluidly connected to thecontrol chamber 100 for directing the microbiological sample to therespective outlet ports.

The diluted sample is fed through an inlet coupler 120 into thedistribution system. Air for one control port is sent through the aircoupler 122 and air for the other control port is sent through thecoupler 124. The diluted sample which is not distributed is dischargedthrough the coupler 126. Initially, the diluted sample is sent inthrough 120 with the air pressure through 124 being greater than the airpressure through 122. In this way the diluted sample will pass seriallyfrom one stage to the next stage and fill all three parallel paths untilthe fluid continues through the discharge coupler 126. The portionfilled by the fluid is shown with dots for clarity. When the dilutedsample completely fills the main paths of the fluid amplifiers, thepressure of the air is switched whereby the pressure is greater throughthe coupler 122 whereby the diluted sample from each stage of theamplifier will be sent to the opposite outlet ports and be dischargedinto the receiving well therebeneath. As heretofore explained, asubstantially identical amount of fluid will be passed out of each stagewhereby a substantially identical amount of the diluted sample will bereceived by each of the wells.

In addition to the distribution system shown built into the cover, thedilution system heretofore described could also be built into the coveras an upper layer or as an adjacent section. In this way, an initialsample can be fed into the cover portion of the Petri dish together withdiluent and directly within the cover portion the sample will beserially diluted and appropriately distributed to receiving wells in thelower portion of the Petri dish. The size of the Petri dish can bemaintained small and the entire dilution and distribution process isautomated and achived in a succeedingly fast time.

The entire Petri dish can be connected to a holder with appropriatelymaintained sterile valve connections for feeding the sample, diluent,and air, etc., into the Petri dish. The feeding of successive Petridishes can be automatic into the holding mechanism and as each Petridish is fed into the holder the appropriate sample and diluent are fedinto the cover portion and the air appropriately controlled for theproper dilution and distribution. After complete distribution of thediluted sample to the receiving wells, the Petri dish is automaticallyremoved and a subsequent dish placed into the holder. The necessarysterility can be maintained throughout. Where different samples areintroduced for each Petri dish, appropriate sterilization of thenecessary valves can be carried out between successive operations.

A further aspect of the microbiological system for automation andminiaturization for a microbiological laboratory concerns the concept oflinear streaking. Heretofore in prior art microbiological use, it hasbeen common practice to provide a serpentine streaking of a sample on aPetri dish in order to achieve proper progressive dilutions of thesample on the growth medium. Typically, a microbiological sample will bediluted and by means of a loop a quantity of sample will be held and theloop will be streaked across a circular Petri dish. The streaking bringsabout the progressive dilutions of the sample on the growth medium.

Even when automating the microbiological laboratory, heretoforeautomatic equipment has been provided which will automatically providethe serpentine streaking of the dish. These automated equipment havebeen expensive and difficult to control and accordingly, have not beenwidely utilized.

The present invention provides the concept of linear streaking in placeof the heretofore accepted serpentine streaking. Thus, an elongatedPetri dish can be provided and an appropriate sample linearly appliedonto the elongated Petri dish. The Petri dishes can be placed in asequence whereby a series of applications of the sample are applied insequence onto the dishes.

Referring now to FIG. 5, there is shown three elongated Petri dishes insequence, the first of which is designated as 130. Viewing the dishesfrom above, there is a base plate 132 on which is provided a growthmedium 134. The subsequent dishes 136 and 138 can be interconnected orspaced apart from each other. The microbiological sample would beapplied linearly across each of the three elongated Petri dishes.

In one variation, a microbiological sample either directly or diluted,will be applied respectively to all of the Petri dishes. However, eachelongated Petri dish will have a different type of growth medium. Withthe same sample applied to various growth mediums, it will be possibleto determine the type, content, etc. of the sample viewing its reactionto the various growth mediums.

In another variation, each of the elongated Petri dishes would have thesame type of growth medium. An applicator would be utilized whichcontinuously diluted the microbiological sample whereby a differentconcentration of the microbiological sample is provided successively tothe various elongated trays.

After the elongated trays or Petri dishes have received the appropriatemicrobiological sample, the trays can be arranged in a desired pattern.For example, as shown in FIG. 6, each tray 140 has a coupling connector142 at one end thereof. The coupling connector can interconnect to thenext adjacent tray 144. All of the trays are thereby interconnected inserpentine fashion and can be contained within a single area 146. Thus,effectively, by utilizing linear streaking with short strokes onelongated trays, the serpentine arrangement can be simulated not byproviding a serpentine streak on a Petri dish, but providing linearsegments and then arranging the trays in a serpentine fashion.

Of course, the elongated trays need not be arranged in serpentinefashion but can be placed serially, in parallel or arranged in any otherpattern. Furthermore, the trays need not necessarily be elongated butcan be square, or other shape, as long as they can receive a linearapplication of the microbiological sample.

Referring now to FIG. 7, there is shown a simple, schematic of anapparatus for linearly applying the microbiological sample onto trays.Initially the trays are stacked at 150. The trays can be eitherindividually placed in a sterile condition or can be provided with aplurality of sterile trays contained in a single package. However, fromtheir stacked arrangement, each of the trays are individually fed to anapplication stage 152 in which the microbiological sample is linearlyapplied to the trays.

The movement is shown by means of a conveyor belt 154 having graspingpins 156 which matingly grasp each tray and move it through successivestages. A first motor M1 moves a first section of the conveyor belt anda second motor M2 moves a second section 158 of the conveyor belt whichremoves the trays from the application stage and sends it to a coveringstage 160 where covers are applied.

In the application stage 152 there is shown a spray head 162 forlinearly spraying the trays. The spray head can be maintained stationarywith the trays providing linear movement therebeneath. Alternately, thetrays can be held in place or moved slowly and the spray head can alsolinearly spray the medium onto the trays.

The spray head 162 can be the output of a serial dilution system wherebyeach successive tray receives a different concentration of themicrobiological sample. One such serial dilution system which can feedthe spray head is of the type heretofore described utilizing fluidamplifiers. Another serial dilution system is of the type which will bedescribed in connection with FIG. 11. A further type of serial dilutionsystem can include a central chamber with radially extending outlet armsin the form of a central hub with outwardly extending spokes. The sampleis initially fed into the central chamber and at each spoke a successiveamount of diluent is fed. The distal ends of each outlet arm approachesthe spray head and provides successive increased dilution of the sample.Other types of serial dilution devices can also be utilized. However,with those heretofore described there can be an exact control of thedilution in order to achieve a desired amount.

Specifically, with heretofore designed serpentine streakingarrangements, the exact concentration at any particular point in theserpentine streak was not directly known nor could it be reproduced.With the present linear streaking, at each linear section there can beprovided a controlled dilution which can be measurably reproduced andcan be predetermined and calculated. For example, any desired geometricprogression of serial dilutions can be achieved. A logarithmic dilutioncan be provided over a very short area with the exact concentration ofthe sample at each section being specifically known and calculatable. Ofcourse, continuous dilutions could also be used.

Referring again to FIG. 7, each of the trays move along the conveyorbelt and receive a linear application of the medium from the spray heador other applicator. The trays move onto the next conveyor section 158where they move under the covering section 160 where covers 162 areplaced on each tray. The trays can then be placed in any of thepatterns, as was described in connection with FIG. 6 and can then be fedinto an incubator 164.

One arrangement of the trays which can be utilized in conjunction withthe system described is shown in FIGS. 8 and 9. The tray itself includesa base 166 on which is placed the growth medium 168. The covers includean upper flat plate 170 with downwardly depending flanges 172 along theperiphery. The side walls 174 of the base are supported by the covermeans by means of inwardly extending struts 176. The walls 174 fit intoreceiving seats 178 formed on the base 166.

Thus, the tray itself only includes the base plate with the growthmedium formed directly thereon. These base plates pass beneath theapplicator for linear application of the microbiological sample. Then,they receive the covers with the downwardly depending sidewalls wherebythe side walls fit into receiving seats on the base plate and a petridish is formed. It should be appreciated that the struts are spacedapart from each other and permit an indirect flow of air therebetween toreach the growth medium to provide the necessary flow of air needed forthe incubation.

At the same time, because of the top plate there is sufficientsturdiness to permit stacking of the elongated Petri dishes after theyhave been innoculated by means of the microbiological sample.

It should further be appreciated that although initially only a lineartray is provided with a growth medium thereon, after application of thecover which fits onto the base plate there is formed a completeconventional Petri dish having the necessary indirect flow of air.

In addition to using a Petri dish with one section, it is possible tohave a plurality of sections in parallel relationship on a single suchPetri dish. Referring now to FIG. 10, there is shown a portion of suchPetri dish generally designated as 180 and including a base plate 182with at least a rear wall 184 and longitudinally extending walls 186 and188 at the respective ends as well as intermediate walls 190 to separatethe dish into a plurality of sections 192. A growth medium would beplaced in each of the sections 192. The intermediate walls includepassageways 194 which can receive a laminar flow of fluid therethroughto maintain each of the longitudinal sections sterile from the nextadjacent section.

Utilizing the Petri dish of the type shown in FIG. 10, it is possible tosimultaneously apply a microbiological sample along a plurality ofchannels within the same dish. The sample would be linearly appliedacross the individual longitudinal sections and by means of the laminarflow of fluid cross contamination between the adjacent sections would beprevented. Each adjacent section could contain a different ingredient ofgrowth medium. Alternately, a common growth medium can be used in all ofthe growth sections and a different concentration of sample would beapplied to each longitudinal section.

Referring now to FIG. 11, there is shown another embodiment of a devicefor providing serial dilutions. The device shown generally at 200includes a cylinder 202 with a piston 204 movable within the cylinder. Arod 206 is coupled to the piston and extends through the top wall 208 ofthe cylinder. The rod is utilized for reciprocally operating the pistonin the cylinder.

An outlet 210, shown as a spray outlet is provided in the bottom 212 ofthe cylinder. A first inlet 214 is located intermediate the lower wall212 and the piston 214 for receiving a microbiological sample. A secondinlet 216 is provided on the upper wall 208 which can receive diluent. Aone-way passage 218 is provided in the side wall of the cylinderpermitting passage from the upper side of the piston to the lower sideof the piston. The one way passage is controlled by means of valves 220and 222 and the passageway 216 is controlled by the valve 224. Inoperation, the microbiological sample is fed through the inlet 214 anddiluent is continuously provided available through the inlet 216. As therod reciprocates, serial dilution will occur.

Specifically, as the rod is lowered, the piston moves downward and aportion of the sample contained in the lower chamber 226 is sprayed outthrough the outlet 210. As the piston moves upward, a portion of thediluent contained in the upper chamber 228 of the cylinder passesthrough the one-way passageway 218 into the lower chamber 226 to mixwith the sample and dilute the sample. On the next downward stroke ofthe piston, a portion of the diluted sample will flow out of the sprayoutlet 210.

On the subsequent upward stroke of the piston, again a portion of thediluent contained in the upper chamber 228 will pass through the one-waypassage 218 and now the previously diluted sample in the lower chamberwill be further diluted by the additional diluent. Again, the downwardstroke will force the now higher diluted sample out of the spray outlet.

In this manner, the continuous reciprocating motion of the pistonprovides serial dilutions of the sample.

In order to facilitate the continued supply of diluent, and provide afree standing unit, a second chamber 230 is interconnected to thecylinder by means of the passageway 232. Initially, the adjacent chamber230 is filled with diluent. Alternately, an inlet 23' can be provided inthe adjacent chamber and diluent inputted to the adjacent chamber. Witheach downward stroke of the piston, additional diluent is fed from theadjacent chamber 230 into the top chamber 228 of the cylinder to providea continued supply of diluent for the serial dilutions.

The top end of the rod 206 can be threaded at 234 and a nut 236 placedappropriately along the threaded portion to control the downward strokeof the piston to thereby control the amount of diluted sample sprayedout of the device.

Each of the foregoing devices and apparatus, as well as the methodsdescribed, all form a part of a total microbiological system which canbe integrated and utilized for the miniaturization and automation of amicrobiological laboratory. At the same time, each device, apparatus,and/or method can be utilized individually to provide improved operationof at least that portion of the total system as is desired.

There has been disclosed heretofore the best embodiment of the inventionpresently contemplated. However, it is to be understood that variouschanges and modifications may be made thereto without departing from thespirit of the invention.

I claim:
 1. A microbiological mixing and dilution device comprising, atleast one fluid amplifier having a control chamber, a first inlet portfor feeding a microbiological fluid sample from a source of supply intosaid control chamber, a second inlet port for feeding a fluid diluentfrom another source of supply into said control chamber wherein thefluid sample and the fluid diluent are mixed, two fluid outlet ports forselectively discharging the contents of said control chamber, saidcontrol chamber serving as a mixing chamber to dilute the sample andcontrol port means fluidly coupled to said control chamber for utilizinga suitable control fluid to direct at least a portion of the dilutedsample to a selected outlet port, and distribution means for receivingthe diluted sample from said selected outlet port for microbiologicaluse.
 2. A microbiological device as in claim 1, further comprising meanscoupled to said control port means for initially directing all of thediluted sample to one outlet port and subsequently switching the sampleto the other outlet port whereby a constant quantity of the dilutedsample is discharged to a receiving well.
 3. A microbiological dilutiondevice comprising:a fluid amplifier having a mixing chamber, an inletport for feeding an aqueous microbiological liquid into said mixingchamber, two fluid outlet ports for selectively discharging the contentsof said mixing chamber, and control port means fluidly coupled to saidmixing chamber for utilizing a suitable control fluid to direct at leasta portion of the aqueous microbiological liquid to said selected outletport; two input means fluidly coupled to said inlet port; supply meansfor providing the aqueous microbiological liquid in the form of amicrobiological sample to one input means and a diluent to the otherinput means for dilution of the sample in said mixing chamber, andoutput means for removing a predetermined volume of the diluted samplefrom one of said outlet ports and the remainder of the diluted samplefrom the other said outlet ports.
 4. A microbiological dilution deviceas in claim 3 further comprising a plurality of fluid amplifiers eachhaving two input means, said amplifiers being serially interconnectedwith a first one of said input means of each of said amplifiers beingfluidly coupled to a first one of said outlet ports of a precedingamplifier, said supply means fluidly coupling the microbiological sampleto a first one of said input means of only the first of said pluralityof amplifiers and the diluent to the second one of said input means ofeach of said amplifiers, and said output means removing saidpredetermined volume of the diluted sample from said second one of saidoutlet ports of each of said amplifiers, whereby each fluid amplifierprovides a further dilution of the sample which it receives from thepreceeding amplifier to thereby achieve a serial dilution of the sample.5. A microbiological dilution device as in claim 4, wherein said controlport means individually controls the selected portion output of each ofsaid amplifiers to thereby control the percent dilution of each portion.6. A microbiological dilution device as in claim 3 further comprisingbaffle means in said mixing chamber to increase the mixing of the samplewith the diluent.
 7. A microbiological distribution device comprising: aplurality of serially connected fluid amplifiers each having a controlchamber, an inlet port for feeding an aqueous microbiological liquidinto said control chamber, first and second fluid outlet ports forselectively discharging the contents of said control chamber, andcontrol port means fluidly coupled to said control chamber, fordetermining the flow of fluid into the respective outlet ports, one ofsaid outlet ports of each of said amplifiers being fluidly coupled tosaid inlet port of said next successive amplifier, said other outletport of each said amplifiers discharging into an array of receivingwells, said inlet port of the first of said amplifiers permitting fluidcoupling to a source of an aqueous microbiological sample fordistribution, selector control means simultaneously coupled to all ofsaid control port means for initially directing all of the aqueousmicrobiological liquid to said first outlet port of each of saidamplifiers and upon actuation said selector control means willsimultaneously direct the aqueous microbiological liquid in eachamplifier to said second outlet port, whereby the aqueousmicrobiological liquid will serially flow through all of said amplifiersand after all of said amplifiers are filled the selector control meansis actuated and the aqueous microbiological liquid will switch to saidsecond outlet port, whereby a substantially equal volume of aqueousmicrobiological liquid will be discharged from each of said amplifiersinto the receiving wells.
 8. A microbiological distribution devices asin claim 7 further comprising detecting means for detecting when allsaid amplifiers have been filled with the working liquid, said selectorcontrol means being responsive to said detecting means for switching theliquid to said other outlet ports.
 9. A microbiological distributiondevice as in claim 7 further comprising a microbiological tray having abase portion and a cover portion, an array of receiving wells formed insaid base portion, said plurality of serially connected fluid amplifiersbeing formed in said cover portion with said other outlet ports of eachamplifier being positioned over one of the receiving wells.
 10. Amicrobiological distribution device as in claim 9, wherein said coverportion comprises an inlet coupler for connection to said supply means,control couplers for connection to said selector control means, and adischarge coupler connected to said one outlet port of the final one ofsaid plurality of amplifiers.
 11. A serial dilution microbiologicaldevice comprising: a cylinder;a piston reciprocally movable in saidcylinder and separating said cylinder into first and second chambers oneon each side of the piston, the reciprocating of the piston within thecylinder changing the volume of the chambers inversely with respect toeach other, said piston preventing fluid flow therethrough between thechambers, a rod coupled to said piston for reciprocally operating saidpiston within said cylinder, an output from said first chamber in saidcylinder, an inlet into said first chamber for receiving amicrobiological sample into said first chamber, an inlet into saidsecond chamber for receiving diluent into said second chamber, and aone-way pasage between said first and said second chambers permittingflow only from said second chamber into said first chamber, wherebyreciprocation of said piston provides a serially diluted sample at theoutput of said cylinder.
 12. A microbiological device as in claim 11further comprising a diluent reservoir and another one-way passagefluidly coupled between said reservoir and said inlet of said secondchamber for flow of diluent into said second chamber.
 13. Amicrobiological device as in claim 11 further comprising limiting meanscoupled to said rod for determining the length of the stroke of saidpiston within said cylinder.